Expression of virulence factors by opportunistic pathogens is controlled, in part, by the environment of the host. The molecular basis of this regulation often involves an interplay of transcriptional factors that modulate expression of virulence genes. This proposed study will examine the mechanism which regulates the expression of selected virulence factors (pilin, flagellin and a newly identified adhesin) that share a requirement for RpoN, the alternative sigma subunit of RNA polymerase. The expression of the pilin gene further depends on the action of two additional regulatory genes, pilR and pilS, that share similarities with members of the paired sensor/regulator family. The transcriptional activator PilR will be purified, and its binding site at the pilin gene will be identified by biochemical and genetic methods. The second protein, PilS, which is a candidate for a protein kinase with PilR as its substrate, will also be characterized to determine whether pilin gene transcription involves phosphorylation of PilS and transfer of phosphate to PilR. The region downstream of the rpoN gene encodes two proteins that are candidates for regulatory elements involved in the repression of genes that are transcribed by RpoN-containing RNA polymerase. To assess the role of these proteins in gene expression, purified preparations will be examined for protein-protein interactions with RpoN, or for binding to RpoN dependent promoter regions. Flagellin expression is dependent on RpoN indirectly, since RpoN controls expression of one or several regulators required for the expression of the flagellin gene. These regulatory factors will be identified and characterized. Recent identification of an RpoN-controlled adhesin will be continued, including identification of the structural and regulatory elements that mediate bacterial adherence to eukaryotic cells. It is hoped that results of work based on this proposal will provide insights on the regulatory network employed by microorganisms causing opportunistic infections, in controlling expression of genes important during the various stages of the pathogenic process.